Extracellular microRNAs (miRs) in endothelial cells (ECs) can be transported by extracellular carriers to regulate the gene expression in the recipient cells, i.e., smooth muscle cells (SMCs) and macrophages (MOs), and hence modulate vascular physiology and pathophysiology. In the current funding cycle, we demonstrated that distinct flow patterns, i.e. pulsatile shear (PS, the main feature of atheroprotective flow) and oscillatory shear (OS, the main feature of atheroprone flow) differentially regulate the miR expression profiles (miRomes) in ECs to cause up- or down-regulation of genes involved in inflammation, redox state, and proliferation. Moreover, others and we have shown that the shear stress-modulated miRs in ECs (e.g., miR-126 and miR- 143/145) are transferable via carriers such as exosomes or Ago2 to SMCs to target their gene expression and alter their phenotype. In relation to translational application, patients with cardiovascular diseases have been shown to have elevations of miR-126 and miR-92a in association with extracellular vesicles, and a decrease of their associations with lipoproteins. These findings led to the hypothesis of this new proposal that atheroprotective and atheroprone flow patterns modulate distinct miR-transportomes and regulate different miR-targetomes to result in beneficial or detrimental outcome of the vasculature. Specifically, we will expand our study to investigate the mechanisms and functional consequences of the shear stress-regulated EC miR transportomes and SMC/MO targetomes. The four Specific Aims are: (1) to investigate the EC miR transportomes regulated by PS vs. OS. (2) To elucidate the miR uptake mechanism and miR targetomes in recipient SMCs and MOs under PS and OS. (3) To determine the roles of PS- and OS-modulated miR transportomes and targetomes in regulating vascular functions. (4) To validate the flow regulation of miR expression and transmission in the vasculature in animal studies in vivo and in clinical samples from patients with cardiovascular diseases. We will perform flow channel experiments, high-throughput screening, and systems biology analysis and modeling to elucidate the diverse transportomes regulated by PS vs. OS in modulating the targetomes and functions of SMCs and MOs. We will validate and translate our in vitro and in silico results by using mouse models as well as human clinical samples. The proposed research aims at investigating the mechanisms by which miRs mediate the flow-regulation of vascular phenotypes and functions, with the ultimate goal of elucidating the interplays of miRs and shear pattern in the regulation of vascular homeostasis for translational applications.